WIPI2b and Atg16L1: setting the stage for autophagosome formation

The double-membraned autophagosome organelle is an integral part of autophagy, a process that recycles cellular components by non-selectively engulfing and delivering them to lysosomes where they are digested. Release of metabolites from this process is involved in cellular energy homoeostasis under basal conditions and during nutrient starvation. Selective engulfment of protein aggregates and dysfunctional organelles by autophagosomes also prevents disruption of cellular metabolism. Autophagosome formation in animals is crucially dependent on the unique conjugation of a group of ubiquitin-like proteins in the microtubule-associated proteins 1A/1B light chain 3 (LC3) family to the headgroup of phosphatidylethanolamine (PE) lipids. LC3 lipidation requires a cascade of ubiquitin-like ligase and conjugation enzymes. The present review describes recent progress and discovery of the direct interaction between the PtdIns3P effector WIPI2b and autophagy-related protein 16-like 1 (Atg16L1), a component of the LC3-conjugation complex. This interaction makes the link between endoplasmic reticulum (ER)-localized production of PtdIns3P, triggered by the autophagy regulatory network, and recruitment of the LC3-conjugation complex crucial for autophagosome formation.

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Scanning Electron-Assisted Dielectric Microscopy Reveals Autophagosome Formation by LC3 and ATG12 in Cultured Mammalian Cells

International Journal of Molecular Sciences ◽

10.3390/ijms22041834 ◽

2021 ◽

Vol 22 (4) ◽

pp. 1834

Author(s):

Tomoko Okada ◽

Toshihiko Ogura

Keyword(s):

Mammalian Cells ◽

Culture Medium ◽

Related Gene ◽

Autophagosome Formation ◽

Intact Cells ◽

Microtubule Associated Proteins ◽

Associated Proteins ◽

The Difference ◽

Related Proteins ◽

Scanning Electron

Autophagy is an intracellular self-devouring system that plays a central role in cellular recycling. The formation of functional autophagosomes depends on several autophagy-related proteins, including the microtubule-associated proteins 1A/1B light chain 3 (LC3) and the conserved autophagy-related gene 12 (Atg12). We have recently developed a novel scanning electron-assisted dielectric microscope (SE-ADM) for nanoscale observations of intact cells. Here, we used the SE-ADM system to observe LC3- and Atg12-containing autophagosomes in cells labelled in the culture medium with antibodies conjugated to colloidal gold particles. We observed that, during autophagosome formation, Atg12 localized along the actin meshwork structure, whereas LC3 formed arcuate or circular alignments. Our system also showed a difference in the distribution of LC3 and Atg12; Atg12 was broadly distributed while LC3 was more localized. The difference in the spatial distribution demonstrated by our system explains the difference in the size of fluorescent spots due to the fluorescently labelled antibodies observed using optical microscopy. The direct SE-ADM observation of cells should thus be effective in analyses of autophagosome formation.

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p62 targeting to the autophagosome formation site requires self-oligomerization but not LC3 binding

The Journal of Cell Biology ◽

10.1083/jcb.201009067 ◽

2011 ◽

Vol 192 (1) ◽

pp. 17-27 ◽

Cited By ~ 252

Author(s):

Eisuke Itakura ◽

Noboru Mizushima

Keyword(s):

Endoplasmic Reticulum ◽

Degradation Process ◽

Related Protein ◽

Autophagosome Formation ◽

Intracellular Degradation ◽

Cargo Receptor ◽

Specific Proteins

Autophagy is an intracellular degradation process by which cytoplasmic contents are degraded in the lysosome. In addition to nonselective engulfment of cytoplasmic materials, the autophagosomal membrane can selectively recognize specific proteins and organelles. It is generally believed that the major selective substrate (or cargo receptor) p62 is recruited to the autophagosomal membrane through interaction with LC3. In this study, we analyzed loading of p62 and its related protein NBR1 and found that they localize to the endoplasmic reticulum (ER)–associated autophagosome formation site independently of LC3 localization to membranes. p62 colocalizes with upstream autophagy factors such as ULK1 and VMP1 even when autophagosome formation is blocked by wortmannin or FIP200 knockout. Self-oligomerization of p62 is essential for its localization to the autophagosome formation site. These results suggest that p62 localizes to the autophagosome formation site on the ER, where autophagosomes are nucleated. This process is similar to the yeast cytoplasm to vacuole targeting pathway.

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A molecular mechanism for salt stress-induced microtubule array formation in Arabidopsis

10.1101/431031 ◽

2018 ◽

Cited By ~ 1

Author(s):

Christopher Kesten ◽

Arndt Wallmann ◽

René Schneider ◽

Heather E. McFarlane ◽

Anne Diehl ◽

...

Keyword(s):

Salt Stress ◽

Molecular Mechanism ◽

Plant Cells ◽

Binding Motif ◽

Related Protein ◽

Microtubule Associated Proteins ◽

Protein Tau ◽

Intrinsically Disordered ◽

Associated Proteins ◽

Hydrophobic Binding

AbstractMicrotubules are filamentous structures necessary for cell division, motility and morphology, with dynamics critically regulated by microtubule-associated proteins (MAPs). We outline the molecular mechanism by which the MAP, COMPANION OF CELLULOSE SYNTHASE1 (CC1), controls microtubule bundling and dynamics to sustain plant growth under salt stress. CC1 contains an intrinsically disordered N-terminus that links microtubules at evenly distributed distances through four conserved hydrophobic regions. NMR analyses revealed that two neighboring residues in the first hydrophobic binding motif are crucial for the microtubule interaction, which we confirmed through live cell analyses. The microtubule-binding mechanism of CC1 is remarkably similar to that of the prominent neuropathology-related protein Tau, indicating evolutionary convergence of MAP functions across animal and plant cells.

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Interactions among p22, glyceraldehyde-3-phosphate dehydrogenase and microtubules

Biochemical Journal ◽

10.1042/bj20040622 ◽

2004 ◽

Vol 384 (2) ◽

pp. 327-336 ◽

Cited By ~ 43

Author(s):

Josefa ANDRADE ◽

Sandy Timm PEARCE ◽

Hu ZHAO ◽

Margarida BARROSO

Keyword(s):

Binding Protein ◽

Direct Interaction ◽

Independent Manner ◽

Microtubule Associated Proteins ◽

Binding Partners ◽

Ef Hand ◽

Associated Proteins ◽

Microsomal Membranes ◽

Terminal Amino

Previously, we have shown that p22, an EF-hand Ca2+-binding protein, interacts indirectly with microtubules in an N-myristoylation-dependent and Ca2+-independent manner. In the present study, we report that N-myristoylated p22 interacts with several microtubule-associated proteins within the 30–100 kDa range using overlay blots of microtubule pellets containing cytosolic proteins. One of those p22-binding partners, a 35–40 kDa microtubule-binding protein, has been identified by MS as GAPDH (glyceraldehyde-3-phosphate dehydrogenase). Several lines of evidence suggest a functional relationship between GAPDH and p22. First, endogenous p22 interacts with GAPDH by immunoprecipitation. Secondly, p22 and GAPDH align along microtubule tracks in analogous punctate structures in BHK cells. Thirdly, GAPDH facilitates the p22-dependent interactions between microtubules and microsomal membranes, by increasing the ability of p22 to bind microtubules but not membranes. We have also shown a direct interaction between N-myristoylated p22 and GAPDH in vitro with a KD of ∼0.5 μM. The removal of either the N-myristoyl group or the last six C-terminal amino acids abolishes the binding of p22 to GAPDH and reduces the ability of p22 to associate with microtubules. In summary, we report that GAPDH is involved in the ability of p22 to facilitate microtubule–membrane interactions by affecting the p22–microtubule, but not the p22–membrane, association.

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Human CLASP2 specifically regulates microtubule catastrophe and rescue

Molecular Biology of the Cell ◽

10.1091/mbc.e18-01-0016 ◽

2018 ◽

Vol 29 (10) ◽

pp. 1168-1177 ◽

Cited By ~ 25

Author(s):

Elizabeth J. Lawrence ◽

Göker Arpag˘ ◽

Stephen R. Norris ◽

Marija Zanic

Keyword(s):

Growth Rates ◽

Molecular Mechanisms ◽

Direct Interaction ◽

Microtubule Dynamics ◽

Microtubule Associated Proteins ◽

Cellular Processes ◽

Associated Proteins ◽

Microtubule Growth ◽

Protein Components

Cytoplasmic linker-associated proteins (CLASPs) are microtubule-associated proteins essential for microtubule regulation in many cellular processes. However, the molecular mechanisms underlying CLASP activity are not understood. Here, we use purified protein components and total internal reflection fluorescence microscopy to investigate the effects of human CLASP2 on microtubule dynamics in vitro. We demonstrate that CLASP2 suppresses microtubule catastrophe and promotes rescue without affecting the rates of microtubule growth or shrinkage. Strikingly, when CLASP2 is combined with EB1, a known binding partner, the effects on microtubule dynamics are strongly enhanced. We show that synergy between CLASP2 and EB1 is dependent on a direct interaction, since a truncated EB1 protein that lacks the CLASP2-binding domain does not enhance CLASP2 activity. Further, we find that EB1 targets CLASP2 to microtubules and increases the dwell time of CLASP2 at microtubule tips. Although the temporally averaged microtubule growth rates are unaffected by CLASP2, we find that microtubules grown with CLASP2 display greater variability in growth rates. Our results provide insight into the regulation of microtubule dynamics by CLASP proteins and highlight the importance of the functional interplay between regulatory proteins at dynamic microtubule ends.

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Luminal particles within cellular microtubules

The Journal of Cell Biology ◽

10.1083/jcb.200606074 ◽

2006 ◽

Vol 174 (6) ◽

pp. 759-765 ◽

Cited By ~ 74

Author(s):

Boyan K. Garvalov ◽

Benoît Zuber ◽

Cédric Bouchet-Marquis ◽

Mikhail Kudryashev ◽

Manuela Gruska ◽

...

Keyword(s):

Mammalian Cells ◽

Three Dimensional ◽

Dimensional Structure ◽

Cross Sectional ◽

Microtubule Associated Proteins ◽

Associated Proteins ◽

Quantitative Manner ◽

Internal Side ◽

20 Nm ◽

Cellular Components

The regulation of microtubule dynamics is attributed to microtubule-associated proteins that bind to the microtubule outer surface, but little is known about cellular components that may associate with the internal side of microtubules. We used cryoelectron tomography to investigate in a quantitative manner the three dimensional structure of microtubules in intact mammalian cells. We show that the lumen of microtubules in this native state is filled with discrete, globular particles with a diameter of 7 nm and spacings between 8 and 20 nm in neuronal cells. Cross-sectional views of microtubules confirm the presence of luminal material in vitreous sections of brain tissue. Most of the luminal particles had connections to the microtubule wall, as revealed in tomograms. A higher accumulation of particles was seen near the retracting plus ends of microtubules. The luminal particles were abundant in neurons, but were also observed in other cells, such as astrocytes and stem cells.

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Towards an understanding of microtubule function and cell organization: an overview

Biochemistry and Cell Biology ◽

10.1139/o92-131 ◽

1992 ◽

Vol 70 (10-11) ◽

pp. 835-841 ◽

Cited By ~ 46

Author(s):

Thomas H. MacRae

Keyword(s):

Dynamic Instability ◽

Functional Integration ◽

Structural Protein ◽

Microtubule Associated Proteins ◽

Microtubule Stability ◽

Microtubule Polarity ◽

Tubulin Isoforms ◽

Associated Proteins ◽

Cell Organization ◽

Cellular Components

Microtubules exhibit dynamic instability, converting abruptly between assembly and disassembly with continued growth dependent on the presence of a tubulin–GTP cap at the plus end of the organelle. Tubulin, the main structural protein of microtubules, is a heterodimer composed of related polypeptides termed α-tubulin and β-tubulin. Most eukaryotic cells possess several isoforms of the α- and β-tubulins, as well as γ-tubulin, an isoform restricted to the centrosome. The isoforms of tubulin arise either as the products of different genes or by posttranslational processes and their synthesis is subject to regulation. Tubulin isoforms coassemble with one another and isoform composition does not appear to determine whether a microtubule is able to carry out one particular activity or another. However, the posttranslational modification of polymerized tubulin may provide chemical signals which designate microtubules for a certain function. Microtubules interact with proteins called microtubule-associated proteins (MAPs) and they can be divided into two groups. The structural MAPs stimulate tubulin assembly, enhance microtubule stability, and influence the spatial distribution of microtubules within cells. The dynamic MAPs take advantage of microtubule polarity and organization to vectorially translocate cellular components. The interactions between microtubules and MAPs contribute to the structural–functional integration that characterizes eukaryotic cells.Key words: tubulin, microtubules, microtubule-associated proteins.

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ER-Phagy and Its Role in ER Homeostasis in Plants

Plants ◽

10.3390/plants9121771 ◽

2020 ◽

Vol 9 (12) ◽

pp. 1771

Author(s):

Yan Bao ◽

Diane C. Bassham

Keyword(s):

Endoplasmic Reticulum ◽

Er Stress ◽

Stress Responses ◽

Recent Progress ◽

Unfolded Proteins ◽

Cellular Survival ◽

Selective Autophagy ◽

Membrane Bound ◽

Er Homeostasis ◽

Cellular Components

The endoplasmic reticulum (ER) is the largest continuous membrane-bound cellular organelle and plays a central role in the biosynthesis of lipids and proteins and their distribution to other organelles. Autophagy is a conserved process that is required for recycling unwanted cellular components. Recent studies have implicated the ER as a membrane source for the formation of autophagosomes, vesicles that transport material to the vacuole during autophagy. When unfolded proteins accumulate in the ER and/or the ER lipid bilayer is disrupted, a condition known as ER stress results. During ER stress, ER membranes can also be engulfed through autophagy in a process termed ER-phagy. An interplay between ER stress responses and autophagy thus maintains the functions of the ER to allow cellular survival. In this review, we discuss recent progress in understanding ER-phagy in plants, including identification of regulatory factors and selective autophagy receptors. We also identify key unanswered questions in plant ER-phagy for future study.

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Characterization of microtubules by dark-field STEM and replication

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s042482010008506x ◽

1991 ◽

Vol 49 ◽

pp. 152-153

Author(s):

S.B. Andrews ◽

R.D. Leapman ◽

P.E. Gallant ◽

T.S. Reese

Keyword(s):

Protein Interactions ◽

Low Dose ◽

Unit Length ◽

Dark Field ◽

Carbon Films ◽

Microtubule Associated Proteins ◽

Molecular Weights ◽

Freeze Dried ◽

Protein Motors ◽

Associated Proteins

As part of a study on protein interactions involved in microtubule (MT)-based transport, we used the VG HB501 field-emission STEM to obtain low-dose dark-field mass maps of isolated, taxol-stabilized MTs and correlated these micrographs with detailed stereo images from replicas of the same MTs. This approach promises to be useful for determining how protein motors interact with MTs. MTs prepared from bovine and squid brain tubulin were purified and free from microtubule-associated proteins (MAPs). These MTs (0.1-1 mg/ml tubulin) were adsorbed to 3-nm evaporated carbon films supported over Formvar nets on 600-m copper grids. Following adsorption, the grids were washed twice in buffer and then in either distilled water or in isotonic or hypotonic ammonium acetate, blotted, and plunge-frozen in ethane/propane cryogen (ca. -185 C). After cryotransfer into the STEM, specimens were freeze-dried and recooled to ca.-160 C for low-dose (<3000 e/nm2) dark-field mapping. The molecular weights per unit length of MT were determined relative to tobacco mosaic virus standards from elastic scattering intensities. Parallel grids were freeze-dried and rotary shadowed with Pt/C at 14°.

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Filaments and membranes in the mitotic apparatus

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s042482010007638x ◽

1983 ◽

Vol 41 ◽

pp. 544-547

Author(s):

Kent McDonald

Keyword(s):

Intermediate Filaments ◽

Mitotic Spindle ◽

Mitotic Apparatus ◽

Light Microscope Level ◽

Microtubule Associated Proteins ◽

Recent Developments ◽

Associated Proteins ◽

Force Generator ◽

Spindle Function ◽

Antibody Technology

At the light microscope level the recent developments and interest in antibody technology have permitted the localization of certain non-microtubule proteins within the mitotic spindle, e.g., calmodulin, actin, intermediate filaments, protein kinases and various microtubule associated proteins. Also, the use of fluorescent probes like chlorotetracycline suggest the presence of membranes in the spindle. Localization of non-microtubule structures in the spindle at the EM level has been less rewarding. Some mitosis researchers, e.g., Rarer, have maintained that actin is involved in mitosis movements though the bulk of evidence argues against this interpretation. Others suggest that a microtrabecular network such as found in chromatophore granule movement might be a possible force generator but there is little evidence for or against this view. At the level of regulation of spindle function, Harris and more recently Hepler have argued for the importance of studying spindle membranes. Hepler also believes that membranes might play a structural or mechanical role in moving chromosomes.

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